LOCUS       Exported                6254 bp ds-DNA     circular SYN 11-SEP-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6254)
  AUTHORS   M. Tominaga et al
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Oct 7, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6254
                     /organism="synthetic DNA construct"
                     /lab_host="Pichia pastoris"
                     /mol_type="other DNA"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             201..1061
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                     1:  201 ..  269 / #ccffcc / signal sequence
                     2:  270 .. 1061 / #ccffcc
                     Cleavage site after base 269"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori (ColE1)
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /note="color: #31849b"
     promoter        2144..2174
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This forward directional feature has 3 segments:
                     1: 2144 .. 2149 / #ffffff / -35
                     2: 2150 .. 2167 / #ffffff
                     3: 2168 .. 2174 / #ffffff / -10"
     protein_bind    2182..2198
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /note="color: #31849b"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     primer_bind     2239..2274
                     /label=MT1565
                     /note="color: 黒; sequence:
                     TTGCGGCCGCGGATCcttattctactggatcttctc; added: 2024-08-31"
     CDS             2254..2816
                     /codon_start=3
                     /locus_tag="Arg4"
                     /label=KpARG4 (563)
                     /label=Arg4
                     /note="color: #993300"
                     /translation="YSTGSSLMPQKKNPDSLELLRGKSGRCFGALAGFLMSIKSIPSTY
                     NKDMQEDKEPLFDTLITVEHSILIASGVVSTLNIDAERMKNALTMDMLATDLADYLVRR
                     GVPFRETHHISGECVRQAEELNLSGIDQLSLEQLKSIDSRFEADVASTFDFEASVEKRT
                     ATGGTSKTAVLKQLDALNEKLES"
     primer_bind     complement(2825..2850)
                     /label=MT1562
                     /note="color: 黒; sequence: aTCATACATAggatccTCTAGAgtcg;
                     added: 2024-08-31"
     promoter        2841..3077
                     /label=KpPhl1.1
                     /note="This forward directional feature has 6 segments:
                     1: 2841 .. 2845 / #------
                     2: 2846 .. 2875 / #99ccff / phlO
                     3: 2876 .. 2880 / #------
                     4: 2881 .. 2915 / #666699 / AOX1 promoter (-197_-163)
                     5: 2916 .. 2924 / #ff0000 / TATA box
                     6: 2925 .. 3077 / #666699 / AOX1 promoter (-153_-1)"
     CDS             3084..3800
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="This feature has 3 segments:
                     1: 3084 .. 3086 / #05fc14
                     2: 3087 .. 3089 / #05fc14 / 1a
                     3: 3090 .. 3800 / #05fc14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      3878..4124
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    4156..4755
                     /locus_tag="T38473"
                     /label=T38473
                     /note="color: #72f525"
     CDS             complement(4773..5567)
                     /codon_start=1
                     /label=KanMX
                     /note="color: #993366"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(5568..5853)
                     /label=GAP promoter (Ogataea parapolymorpha)
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(5860..5876)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctt gcggccgcgg atccttattc tactggatct tctctgatgc
     2281 ctcaaaaaaa aaacccagac tctttggagt tattgagggg taaatctggt agatgttttg
     2341 gggccttggc tggtttcctc atgtctatta agtccattcc gtcaacctat aacaaagata
     2401 tgcaagagga taaggagcct ttatttgata ctctaatcac tgtagagcac tcgattttga
     2461 tagcatccgg tgtagtttct accttgaaca ttgatgccga acgaatgaag aatgctctaa
     2521 ctatggatat gctggctaca gatcttgccg actatttagt tagaagggga gttccattca
     2581 gagaaactca ccacatttct ggtgaatgtg tcagacaagc cgaggagttg aacctttctg
     2641 gtattgatca gttgtccctc gaacaattga aatccattga ctcccgtttt gaggctgatg
     2701 tggcttcaac gtttgacttt gaagccagtg ttgaaaaaag aactgccacc ggaggaactt
     2761 ctaagactgc tgttttaaag caattggatg cactgaatga aaagctagag tcttgactgc
     2821 aggtcgactc tagaggatcc tatgtatgat acgaaacgta ccgtatcgtt aaggtagcgt
     2881 caaaatttaa ctgttctaac ccctacttga cagcaatata taaacagaag gaagctgccc
     2941 tgtcttaaac cttttttttt atcatcatta ttagcttact ttcataattg cgactggttc
     3001 caattgacaa gcttttgatt ttaacgactt ttaacgacaa cttgagaaga tcaaaaaaca
     3061 actaattatt cgaaacgact agtatggttt ctaagggtga agagttgttc actggtgttg
     3121 ttccaatctt ggttgagttg gacggtgacg ttaacggaca caagttctct gtttctggtg
     3181 aaggtgaggg tgacgctact tacggaaagt tgactttgaa gttcatctgt actactggta
     3241 agttgccagt tccatggcca actttggtta ctactttgac ttacggtgtt cagtgtttct
     3301 ccagataccc agaccacatg aagcagcacg atttcttcaa gtctgctatg ccagagggtt
     3361 acgttcaaga gagaactatc ttcttcaagg acgacggtaa ctacaagact agagctgagg
     3421 ttaagttcga gggtgacaca ttggttaaca gaatcgagtt gaagggtatc gacttcaaag
     3481 aggacggaaa catcttgggt cacaagttgg agtacaacta caactcccac aacgtttaca
     3541 tcatggctga caagcagaag aacggtatca aggttaactt caagatcaga cacaacatcg
     3601 aggacggttc cgttcaattg gctgaccact accaacagaa cactccaatt ggtgacggtc
     3661 cagttttgtt gccagacaac cactacttgt ccactcaatc cgctttgtcc aaggacccaa
     3721 acgagaagag agatcacatg gttttgttgg agttcgttac tgctgctggt atcactttgg
     3781 gtatggacga gttgtacaag taactcgagc cttagacatg actgttcctc agttcaagtt
     3841 gggcacttac gagaagaccg gtcttgctag attctaatca agaggatgtc agaatgccat
     3901 ttgcctgaga gatgcaggct tcatttttga tactttttta tttgtaacct atatagtata
     3961 ggattttttt tgtcattttg tttcttctcg tacgagcttg ctcctgatca gcctatctcg
     4021 cagctgatga atatcttgtg gtaggggttt gggaaaatca ttcgagtttg atgtttttct
     4081 tggtatttcc cactcctctt cagagtacag aagattaagt gagacgttcg tttgtgcaag
     4141 cttagatctt ctagagacaa taagaagaaa aaaaaagaaa agcggtgggg gagggattat
     4201 taaataagga ttatgtaacc ccagggtacc gttctataca tatttaagga ttatttagga
     4261 caatcgatga aatcggcatc aaactggatg ggagtatagt gtccggataa tcggataaat
     4321 catcttgcga ggagccgctt ggttggttgg tgagaggagt gaaatatgtg tctcctcacc
     4381 caagaatcgc gatatcagca ccctgtgggg gacactattg gcctccctcc caaaccttcg
     4441 atgtggtagt gctttattat attgattaca ttgattacat agctaaaccc tgcctggttg
     4501 caagttgagc tccgaattcc aatattagta aaatgcctgc aagataacct cggtatggcg
     4561 tccgaccccg cttaattatt ttaactcctt tccaacgagg acttcgtaat ttttgattag
     4621 ggagttgaga aacggggggt cttgatacct cctcgatttc agatcccacc ccctctcagt
     4681 cccaagtggg acccccctcg gccgtgaaat gcgcgcactt tagttttttt cgcatgtaaa
     4741 cgccggtgtc cgtcatctag agaattcccc gctcagaaga actcgtcaag aaggcgatag
     4801 aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc
     4861 cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg
     4921 tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg
     4981 atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgcgc
     5041 gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca
     5101 tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct
     5161 tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc
     5221 atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact
     5281 tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa
     5341 ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg
     5401 gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac
     5461 acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc
     5521 acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatatg gtttctatat
     5581 tatctttgta ctaaagagca attgataatg tgcgagaaaa actggtcctt atataccgtt
     5641 tgcagcactc cctcccgaac tttacgaaaa gtcgtgcgcc acctgatttt catcacgcca
     5701 aaaacctaca cgtatgacta ctccgggcca gtgttcacca cgagctatat agtgttaatt
     5761 aattacctta ttggttagct ctgcatgtaa gggtggtgtg agccgggaat tgggtctact
     5821 ctagcgttca gtaaggtgat ataaagctct gtagaattca ctggccgtcg ttttacaacg
     5881 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     5941 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     6001 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     6061 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     6121 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     6181 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     6241 accgaaacgc gcga
//
